Hypoxia induces Smad2 phosphorylation signaling and increases stem cell-like properties and multi-drug resistance in K562 leukemic cells

Abstract Elderly AML patients have an unfavorable prognosis with frequent resistance of leukemic cells to chemotherapy. The expression of proteins related with apoptosis and multi-drug resistance have been involved in chemotherapeutic resistance in AML; however, these proteins are not only expressed on leukemic but also on normal cells. Moreover, within leukemic cells, different stages of maturation may occur. The aim of the present study was to evaluate the expression of proteins related to apoptosis (APO 2.7, bax, and bcl-2), and drug resistance (P-gp, MRP, LRP) in a series of 117 elderly AML patients (median age of 70) uniformly treated according with the Spanish Pethema cooperative group protocol (LMA >65). 63 patients (54%) achieved complete remission (CR), with a median relapse free survival (RFS) of 9.2 months. In the bone marrow (BM) pre-treatment, we performed a four-color staining technique (APO2.7, bax or bcl-2 with CD32/CD34/CD45; and P-gp, MRP, or LRP with CD34/CD19/CD33), allowing the identification of myeloid blast cells, and the discrimination between stages of maturation of blast cell subsets. Samples were acquired on a FACSCalibur flow cytometer (Becton/Dickinson), and appropriate controls were used in each individual sample and, accordingly, antigen expression in individual cell samples was quantified as relative fluorescence intensity (RFI) (ratio blast cell/control mean channel fluorescence value). Our results showed that the level of expression of MRP and LRP was significantly higher (mean±SD of MFI 2.0±0.75 vs 2.7±1.87 and 4.5±2.2 vs 7.0±6.2, respectively -p <0.03-) in cases that did not achieved CR with one or two cycles of chemotherapy. Although different patterns of expression of apoptosis or drug resistance-associated proteins were detected according with the stage of maturation on myeloid blast cells, we did’t found any other correlation between levels of bcl-2, bax, APO 2.7, bcl-2/bax ratio or MDR and probability of achieving morphological CR. In addition, CD34 expression on blast cells had also significant influence on response (CR was achieved in 81% of CD34 negative cases vs only 46% of CD34 positive cases; p= 0.005). We didn’t found any influence of apoptosis and/or drug-resistance proteins expression in relapse rate nor RFS. Variables influencing RFS were age (p=0.04), and number of cycles to achieve CR (p=0.01); while only WBC count (p=0.05) influenced overall survival (OS). We conclude that MRP and LRP could have a role in the resistance to induction treatment in elderly AML patients but not in RFS or OS.

Download Full-text

Assessment of effects of multi drug resistance on dielectric properties of K562 leukemic cells using electrorotation

RSC Advances ◽

10.1039/c4ra04873c ◽

2014 ◽

Vol 4 (85) ◽

pp. 44879-44887 ◽

Cited By ~ 14

Author(s):

Garsha Bahrieh ◽

Murat Erdem ◽

Ebru Özgür ◽

Ufuk Gündüz ◽

Haluk Külah

Keyword(s):

Drug Resistance ◽

Multidrug Resistant ◽

Leukemic Cells ◽

Leukemia Cells ◽

Multi Drug Resistance ◽

Human Leukemia ◽

Dielectric Characterization ◽

Human Leukemia Cells ◽

3D Electrodes

In this study, dielectric characterization of multidrug resistant (MDR) K562 human leukemia cells was carried out using a MEMS based electrorotation (ER) device with 3D electrodes.

Download Full-text

Role of ABCG2 in Multi Drug Resistance and Cancer Stem Cell

International Journal of Cell Science & Molecular Biology ◽

10.19080/ijcsmb.2017.03.555619 ◽

2017 ◽

Vol 3 (4) ◽

Author(s):

Siddik Sarkar

Keyword(s):

Drug Resistance ◽

Stem Cell ◽

Cancer Stem Cell ◽

Multi Drug Resistance

Download Full-text

Isolation of cancer stem cells by selection for miR-302 expressing cells

10.1101/427732 ◽

2018 ◽

Author(s):

Karim Rahimi ◽

Annette C. Füchtbauer ◽

Fardin Fathi ◽

Seyed Javad Mowla ◽

Ernst-Martin Füchtbauer

Keyword(s):

Stem Cells ◽

Drug Resistance ◽

Stem Cell ◽

Cancer Stem Cells ◽

Tumor Cells ◽

Host Gene ◽

Multi Drug Resistance ◽

Term Therapy ◽

Specific Gene ◽

Specific Marker

AbstractCancer stem cells receive increasing interest because they are believed to be a major reason for long-term therapy failure. The reason for the therapy resistance of cancer stem cells lies partially in their multi-drug resistance and partially in the ability to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more ‘differentiated’, i.e. less stem cell-like tumor cells. Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. We inserted a selectable egfp-neo coding sequence in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, we used 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. After three weeks, tumors were removed for analysis and primary cultures were established. Stem-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it were not the original ES cells that have been isolated. In conclusion, we show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

Download Full-text

The Nitric Oxide-Generating Prodrug Js-K Reverses the Multi-Drug Resistance Phenotype in AML Cells

Blood ◽

10.1182/blood.v128.22.5127.5127 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5127-5127

Author(s):

Stephan Morys ◽

Catherine Gannage ◽

Moises Terrazas ◽

Imit Kaur ◽

Ken M Kosak ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Cycle ◽

Drug Resistance ◽

Efflux Pump ◽

Leukemic Cells ◽

Multi Drug Resistance ◽

Variable Expression ◽

The Difference

Abstract Nitric oxide (NO) is toxic to acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolates class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. A major cause of treatment failure in AML is the multi-drug resistance (MDR) phenotype associated with overexpression of the P-glycoprotein (Pgp) by leukemic cells. Here, we investigated the effect of JS-K and its formulation on the MDR phenotype in AML cells using HL-60 cells and HL-60/RV+ cells as a model. HL-60/RV+ are selected from the parent HL-60 line and express the Pgp. They are maintained in culture under vincristine (VCR) selection. We studied the effect of the formulation alone (P123), JS-K without the formulation (free JS-K), and JS-K in the formulation (P123/JS-K). Both VCR and free JS-K were cytotoxic towards HL-60 cells with 50% inhibitory concentrations (IC50) of around 0.4 and 0.1 μM, respectively. By contrast, the IC50 for VCR-treated HL-60/RV+ cells, was greater than 1 μM. Free JS-K was cytotoxic to HL-60/RV+ with an IC50 of around 0.2 μM. Pretreatment of HL-60/RV+ cells for 2 hours with free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123 sensitized HL-60/RV+ cells to the effect of VCR: after 3 days of culture, the VCR IC50went from 1.5 μM in control cells to an estimated 1, 0.9, and 0.75 μM for P123, free JS-K, and P123/JS-K pre-treated cells, respectively. We then conducted cell cycle analyses of HL-60/RV+ cells treated with different combinations of JS-K and VCR using propidium idodide staining. Cells were treated with combinations of VCR (1.75 μM), free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123. After 24 hours, the observed percentage (average of 3 different repeats per variable) of the sub-G0/G1 fraction was 7, 6,14, 14, 25, 26, and 43 for untreated controls, free JS-K, P123/JS-K, VCR, free JS-K + VCR, P123 + VCR, and P123/JS-K + VCR, respectively. At 48 hours, the sub-G0/G1 fraction was 4, 5, 5, 19, 20, 23, and 28% for the same variables, respectively. At 72 hours, the sub-G0/G1fraction was 3, 3, 3, 11, 12, 19, and 19% for the same variables, respectively. Thus, the peak effect on the cell cycle was observed at 24 hours. We also observed an effect of P123 alone. In order to determine whether JS-K affects the efflux pump itself, we investigated the effect of 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123 on Rho-123 accumulation in HL-60/RV+ cells after 6 hours of treatment. As a percent of untreated controls, Rho-123 accumulation (average of 9 repeats) was 143, 159, and 144 for free JS-K, P123, and P123/JS-K, respectively (P < 0.05 for all differences with untreated controls). Using flow cytometry, we also determined whether JS-K affects the expression of the Pgp by HL-60/RV+ cells treated with 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123. After 24 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 90, 79, and 63, for P123, free JS-K, and P123/JS-K, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 48 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 83, 79, and 57, for the same variables, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 72 hours, there were no significant differences in the expression of Pgp between the different treatments and untreated controls. We conclude that JS-K in a P123 Pluronic® formulation can reverse the MDR phenotype. The Pluronic® polymers themselves could also affect the MDR phenotype. As such, P123/JS-K could constitute a major addition to our armamentarium for the treatment of AML. Disclosures Shami: JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text